Hypocholesterolemic use of bis(3,5-di-tertiary-butly-4-hydroxyphenylthio)methane

ABSTRACT

The present invention relates to a method of lowering total serum cholesterol in a patient in need thereof comprising administering to said patient a therapeutically effective hypocholesterolemic amount of bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane.

This is a continuation in part of Ser. No. 07/281,433, filed December 8,1988 now U.S. Pat. No. 4,900,757.

Atherosclerosis as manifested in its major clinical complication,ischaemic heart disease, continues to be a major cause of death inindustrialized countries. It is now well accepted that atherosclerosiscan begin with local injury to the arterial endothelium followed byproliferation of arterial smooth muscle cells from the medial layer tothe intimal layer along with deposition of lipid and accumulation offoam cells in the lesion. As the atherosclerotic plaque develops itprogressively occludes more and more of the affected blood vessel andcan eventually lead to ischaemia or infarction. Therefore, it isdesirable to provide methods of inhibiting the progression ofatherosclerosis in patients in need thereof.

There is now a large body of evidence demonstrating thathypercholesterolemia is an important risk factor associated with heartdisease. For example, in December 1984, a National Institute of HealthConsensus Development Conference Panel concluded that loweringdefinitely elevated blood cholesterol levels (specifically blood levelsof low-density lipoprotein cholesterol) will reduce the risk of heartattacks due to coronary heart disease. Accordingly, it is desirable toprovide a method for reducing plasma cholesterol in patients withhypercholesterolemia.

Typically, cholesterol is carried in the blood of warm-blooded animalsin certain lipid-protein complexes such as chylomicrons, very lowdensity lipoprotein (VLDL), low density lipoprotein (LDL), and highdensity lipoprotein (HDL). It is widely accepted that LDL functions in away that directly results in deposition of the LDL cholesterol in theblood-vessel wall and that HDL functions in a way that results in theHDL picking up cholesterol from the vessel wall and transporting it tothe liver where it is metabolized [Brown and Goldstein, Ann. REv.Biochem. 52, 223 (1983); Miller, Ann. Rev. MEd. 31, 97 (1980) . Forexample, in various epidemiologic studies the LDL cholesterol levelscorrelate well with the risk of coronary heart disease whereas the HDLcholesterol levels are inversely associated with coronary heart disease[Patton et al., Clin. Chem. 29, 1890 (1983)]. It is generally acceptedby those skilled in the art that reduction of abnormally high LDLcholesterol levels is effective therapy not only in the treatment ofhypercholesterolemia but also in the treatment of atherosclerosis.Accordingly, it is desirable to provide a method for reducing LDLcholesterol in patients with hypercholesterolemia.

Furthermore, there is evidence based on animal and laboratory findingsthat peroxidation of LDL facilitates the accumulation of cholesterol inmonocyte/macrophages which eventually are transformed into foam cellsand become deposited in the sub-endothelial space of the vessel wall.The accumulation of foam cells in the vessel wall is recognized as anearly event in the formation of an atherosclerotic plaque. Thus it isbelieved that peroxidation of LDL is an important prerequisite to thefacilitated accumulation of cholesterol in the vessel wall and thesubsequent formation of an atherosclerotic plaque. For example, it hasbeen shown that monocyte/macrophages take up and degrade native LDL atrelatively low rates and without marked accumulation of cholesterol. Incontrast, oxidized LDL is taken up by these monocyte/macrophages at muchhigher rates and with marked accumulation of cholesterol [Parthasarathyet al., J. Clin. Invest. 77, 641 (1986)].

It has been shown that2,2'-bis(3,5-di-tertiary-butyl-4hydroxyphenylthio)propane (also known asprobucol), which is a known antioxidant, may prevent the progression ofatherosclerosis in a manner which is independent of its effect onlowering plasma cholesterol levels [See Kita et al. Proc. Natl. Acad.Sci. USA 84, 5298, (1987); Carew et al., Proc. Natl. Acad. Sci. USA 84,7725, (1987)]. It is believed that antioxidants, such as probucol, mayprevent or inhibit the development of atherosclerosis by inhibiting theperoxidation of LDL and thus preventing the facilitated accumulation ofcholesterol in monocyte/macrophages which eventually are transformedinto foam cells and become deposited in the sub-endothelial space of thevessel wall [See Parthasarathy et al. J. Clin. Invest. 77, 641 (1986)].Accordingly, it is desirable to provide a method of inhibiting theperoxidation of LDL.

Barnhart and Shea in U.S. Pat. No. 3,862,332 disclosed that2,2'-bis(3,5-di-tertiary-butyl-4hydroxyphenylthio)propane, also known asprobucol, and 1,1'bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)ethaneare useful in lowering serum cholesterol in animals. Probucol iscurrently approved for administration to man by the U.S. Food and DrugAdministration as a pharmaceutical agent indicated for the reduction ofelevated serum cholesterol in patients with primary hypercholesterolemia(elevated LDL) who have not responded adequately to diet, weightreduction and control of diabetes mellitus (See "Physician's DeskReference", 42nd Edition, 1988, Medical Economics Company, Inc.,Oradell, N.J. 07649). In British Patent Specification No. 1,199,871,probucol was disclosed as providing a 59% reduction in the bloodcholesterol of mice after a 2 week treatment. However, in that samereference, it was disclosed that the compound involved in the method ofuse of the present invention, i.e.,bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane, provided noreduction in the blood cholesterol of mice after comparable treatment.Surprisingly, bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane hasnow been found to be effective in lowering total serum cholesterol, inlowering LDL cholesterol, in inhibiting peroxidation of LDL and ininhibiting the progression of atherosclerosis in patients in needthereof.

For example, bis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methanereduces serum cholesterol and prevents the progression ofatherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit.The WHHL rabbit is a strain of rabbit with a consistently inheritedhyperlipidemic trait. Because these animals lack LDL receptors, theyhave abnormally elevated concentrations of serum cholesterol andspontaneous development of aortic atherosclerosis [see Watanabe,Atherosclerosis 36, 261 (1980)]. WHHL rabbits are well recognized as ananimal model for human familial hypercholesterolemia and it haspreviously been shown [Kita et al., Proc. Natl. Acad. Sci. USA 84, 5928(1987); Carew et al., Proc. Natl. Acad. Sci USA 84, 7725 (1987)]thatprobucol is effective in preventing the progression of atherosclerosisin this model.

In addition, Parthasarathy et al.[J. Clin. Invest. 77, 641 (1986)]showedthat probucol inhibits the oxidative modification of LDL by cupric ionsin vitro and by endothelial cells in vivo. Kita et al. [Proc. Natl.Acad. Sci. USA 84, at 5931]thus theorize that probucol might inhibitatherosclerosis in the WHHL rabbit "by limiting oxidative modificationof LDL and subsequently by limiting foam cell transformation ofmacrophages in vivo".

The present invention relates to the use ofbis(3,5-ditertiary-butyl-4-hydroxyphenylthio)methane in treatingatherosclerosis and/or hypercholesterolemia in patients sufferingtherefrom.

More specifically, the present invention provides a method of loweringtotal serum cholesterol and of lowering LDL cholesterol in a patient inneed thereof comprising administering to said patient a therapeuticallyeffective hypocholesterolemic amount ofbis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane. The presentinvention also provides a method of inhibiting the progression ofatherosclerosis in a patient in need thereof comprising administering tosaid patient a therapeutically effective antiatherosclerotic amount ofbis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane. Finally, thepresent invention provides a method of inhibiting the peroxidation ofLDL in a patient in need thereof comprising administering to saidpatient a therapeutically effective antioxidant amount ofbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane.

As used herein, the term "patient" refers to warmblooded animals ormammals, including WHHL rabbits and humans but not including rodents,who are in need of treatment for atherosclerosis orhypercholesterolemia, such as, for example, in the case of a patientsuffering from familial hyperlipidemia.

Hypercholesterolemia is a disease state characterized by levels of serumcholesterol or of LDL cholesterol which are elevated by a clinciallysignifiant amount over that considered normal by those of ordinary skillin the art. The identification of those patients who are in need oftreatment for hypercholesterolemia is well within the ability andknowledge of one skilled in the art. For example, individuals who haveserum cholesterol levels or LDL cholesterol levels, as determined byclinical laboratory tests, which are substantially and chronicallyelevated over that considered normal by those of ordinary skill in theart, are patients in need of treatment for hypercholesterolemia. By wayof further example, individuals who are at risk of developinghypercholesterolemia can also be patients in need of treatment forhypercholesterolemia. A clinician skilled in the art can readilyidentify, by the use of clinical tests, physical examination andmedical/family history, those patients who are suffering fromhypercholesterolemia and those who are at risk of developinghypercholesterolemia and thus readily determine if an individual is apatient in need of treatment for hypercholesterolemia.

An effective hypocholesterolemic amount ofbis(3,5-ditertiary-butyl-4-hydroxyphenylthio)methane is an amount whichis effective in reducing serum cholesterol levels or LDL cholesterollevels in a patient in need thereof. As such, successful treatment of apatient for hypercholesterolemia is understood to include reducing apatient's serum cholesterol or LDL cholesterol levels. Successfultreatment for hypercholesterolemia is also understood to includeprophylaxis in preventing clinically significant elevations in serumcholesterol or in LDL cholesterol levels in a patient who is at risk ofthe development of hypercholesterolemia.

Atherosclerosis is a disease state characterized by the development andgrowth of atherosclerotic lesions or plaque. The identification of thosepatients who are in need of treatment for atherosclerosis is well withinthe ability and knowledge of one skilled in the art. For example,individuals who are either suffering from clinically significantatherosclerosis or who are at risk of developing clinically significantatherosclerosis are patients in need of treatment for atherosclerosis. Aclinician skilled in the art can readily determine, by the use ofclinical tests, physical examination and medical/family history, if anindividual is a patient in need of treatment for atherosclerosis.

An effective antiatherosclerotic amount ofbis(3,5-ditertiary-butyl-4-hydroxyphenylthio)methane is an amount whichis effective in inhibiting development or growth of atherosclerosis in apatient in need thereof. As such, successful treatment of a patient foratherosclerosis is understood to include effectively slowing,interrupting, arresting, or stopping atherosclerotic lesion or plaquedevelopment or growth and does not necessarily indicate a totalelimination of the atherosclerosis. It is further understood andappreciated by those skilled in the art that successful treatment foratherosclerosis can include prophylaxis in preventing atheroscleroticlesion or plaque formation.

It is believed that peroxidation of LDL facilitates the deposition ofcholesterol in macrophages which subsequently are deposited in thevessel wall and are transformed into foam cells. The identification ofthose patients who are in need of inhibition of peroxidation of LDL iswell within the ability and knowledge of one skilled in the art. Forexample, those individuals who are in need of treatment foratherosclerosis as defined hereinabove, are also patients who are inneed of inhibition of peroxidation of LDL. Furthermore, an effectiveantioxidant amount of bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane is an amount which iseffective in inhibiting the peroxidation of LDL in the patient's blood.

An effective antiatherosclerotic, antioxidant or hypocholesterolemicdose can be readily determined by the use of conventional techniques andby observing results obtained under analogous circumstances. Indetermining the effective dose, a number of factors are consideredincluding, but not limited to: the species of patient; its size, age,and general health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; and the use of concomitantmedication.

An effective antiatherosclerotic, antioxidant or hypocholesterolemicamount of bis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane willgenerally vary from about 1 milligram per kilogram of body weight perday (mg/kg/day) to about 5 grams per kilogram of body weight per day(gm/kg/day). A daily dose of from about 1 mg/kg to about 500 mg/kg ispreferred.

In effecting treatment of a patient, bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane can be administered in anyform or mode which makes the compound bioavailable in effective amounts,including oral and parenteral routes. For example, the compound can beadministered orally, subcutaneously, intramuscularly, intravenously,transdermally, intranasally, rectally, and the like. Oral administrationis generally preferred. One skilled in the art of preparing formulationscan readily select the proper form and mode of administration dependingupon the disease state to be treated, the stage of the disease, andother relevant circumstances.

Bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane can beadministered in the form of pharmaceutical compositions or medicamentswhich are made by combiningbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane withpharmaceutically acceptable carriers or excipients, the proportion andnature of which are determined by the chosen route of administration,and standard pharmaceutical practice.

The pharmaceutical compositions or medicaments are prepared in a mannerwell known in the pharmaceutical art. The carrier or excipient may be asolid, semi-solid, or liquid material which can serve as a vehicle ormedium for the active ingredient. Suitable carriers or excipients arewell known in the art. The pharmaceutical composition may be adapted fororal or parenteral use and may be administered to the patient in theform of tablets, capsules, suppositories, solution, suspensions, or thelike.

The pharmaceutical compositions may be administered orally, for example,with an inert diluent or with an edible carrier. They may be enclosed ingelatin capsules or compressed into tablets. For the purpose of oraltherapeutic administration, bis(3,5-di-tertiary-butyl-4- ().hydroxyphenylthio)methane may be incorporated with excipients and usedin the form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. These preparations should contain atleast 4% of bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane, theactive ingredient, but may be varied depending upon the particular formand may conveniently be between 4% to about 70% of the weight of theunit. The amount of the active ingredient present in compositions issuch that a unit dosage form suitable for administration will beobtained.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders, such as microcrystallinecellulose, gum tragacanth or gelatin; excipients, such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants, such as magnesium stearate or Sterotex;glidants, such as colloidal silicon dioxide; and sweetening agents, suchas sucrose or saccharin may be added or flavoring agents, such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the activeingredient, sucrose as a sweetening agent and certain preservatives,dyes and colorings and flavors. Materials used in preparing thesevarious compositions should be pharmaceutically pure and non-toxic inthe amounts used.

For the purpose of parenteral administration, bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane may be incorporated into asolution or suspension. These preparations should contain at least 0.1%of a compound of the invention, but may be varied to be between 0.1 andabout 50% of the weight thereof. The amount of the active ingredientpresent in such compositions is such that a suitable dosage will beobtained.

The solutions or suspensions may also include one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of toxicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

Bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane can be prepared bymethods well known and appreciated by those of ordinary skill in theart. For example, the compound can be prepared by treating2,6-di-tertiary-butyl4-mercaptophenol with 1,3,5-trioxane in thepresence of acetonitrile and DOWEX 50 resin under reflux conditions.2,6-Di-tertiary-butyl-4-mercaptophenol can be prepared as described, forexample, by Krauss in U.S. Pat. No. 4,734,527.

The following examples illustrate the preparation and use ofbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane according to thepresent invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLE 1 Preparation ofBis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane

Combine acetonitrile [1800 milliliters (ml)], 1,3,5trioxane [71.0 grams(gm), 0.79 moles (mol)], 2,6-ditertiary-butyl-4-mercaptophenol [678.4gm, 2.85 mol]and 2.5 gm DOWEX 50 resin in a three-necked flask with athermowell. Bring the mixture to reflux under a nitrogen 5 atmosphereand maintain for 36-48 hours to provide the title compound.

Filter the mixture to remove the DOWEX 50 resin and concentrate thefiltrate in vacuo to give an amber oil. Dissolve the oil in 1 liter ofethanol at 70 degrees Celsius (°C.) and add 125 ml of water. Allow themixture to cool to ambient temperature over night while stirring.Collect the resulting crystalline product by filtration and wash thefilter cake with 75 ml of cold ethanol/water (90/10). Recrystallize theproduct from ethanol/water and collect by filtration Wash the filtercake with 50 ml of cold ethanol and dry the product in a vacuum oven at50° C and 15 mm Hg overnight to yield 406.9 gm of the purified titlecompound as a white solid. Melting point 94-95° C. Elemental analysis:

30 Calculated--C=71.3%, H=9.07%;

Found--C=71.3%, H=9.09%.

EXAMPLE 2 Inhibition of LDL Peroxidation

Determine the degree of inhibition of LDL peroxidation by probucol andbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane by the method ofYagi et al. [Vitamins 39, 105 (1968)]. Incubate a 0.5 ml solutioncontaining 250 micrograms (μg) of LDL with either probucol orbis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane, in amounts varyingfrom 0 to 30 μg, for 30 minutes at 42° C. To this mixture add 1 ml of a0 cupric sulfate solution (final concentration 12.5 μM) and incubate at37° C. for 2.5 hours. Determine the amount of peroxidation of LDL by thethiobarbituric acid assay. Calculate the dose of probucol andbis(3,5-di-tertiarybutyl-4-hydroxyphenylthio)methane required to inhibit50% of LDL peroxidation (ID₅).

As shown in Table 1, the ID₅₀ for probucol is 12.5 μg whereas the ID₅₀for bis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane is only 6 μg.Therefore, this data indicates thatbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio)methane is over twice aseffective in inhibiting the peroxidation of human LDL than is probucol.

                  TABLE 1                                                         ______________________________________                                        Effect of Probucol and Bis(3,5-di-tertiary-butyl-4-                           hydroxyphenylthio)methane on LDL Peroxidation                                        Treatment.sup.a                                                                       ID.sub.50                                                      ______________________________________                                               A       12.5 μg                                                            B         6 μg                                                      ______________________________________                                         .sup.a Treatment A = Probucol                                                 Treatemnt B = bis(3,5ditertiary-butyl-4-hydroxy phenylthio)methane       

EXAMPLE 3 Hypocholesterolemic Effects ofBis(3,5-di-tertiarybutyl-4-hydroxyphenylthio)methane in Cholesterol-fedRabbits

New Zealand White Rabbits were separated into two groups of 4 or 5rabbits per group. During a Pretreatment Period, one group [ControlGroup]was fed standard rabbit chow for 12 weeks and the other group[Treatment Group]received standard rabbit chow containingbis(3,5-di-tertiarybutyl-4-hydroxyphenylthio)methane (1% by weight) for12 weeks. At the end of the 12 week pretreatment period, the ControlGroup was fed standard rabbit chow containing cholesterol (1% by weight)for 7 weeks, while the Treatment Group received standard rabbit chowcontaining bis (3,5-ditertiarybutyl-4-hydroxyphenylthio)methane (1% byweight) and cholesterol (1% by weight) for 7 weeks. During the TreatmentPeriod, and specifically on days 15, 22, 35 and 49 after starting intothe Treatment Period, blood samples were taken from the animals and wereanalyzed for plasma cholesterol by an enzymatic method using a DACOSanalyzer (Coulter Electronics Inc., Hialeah, Fla., USA).

The results of treatment of cholesterol-fed rabbits withbis(3,5-di-tertiarybutyl-4-hydroxyphenylthio)methane is shown in Table2.

                                      TABLE 2                                     __________________________________________________________________________    Hypocholesterolemic Effects of Bis(3,5-di-                                    tertiarybutyl-4-hydroxyphenylthio)methane in                                  Cholesterol-fed Rabbits                                                             Plasma  Plasma  Plasma  Plasma                                                Cholesterol                                                                           Cholesterol                                                                           Cholesterol                                                                           Cholesterol                                           (mg/dl ± s.d..sup.c)                                                               (mg/dl ± s.d.)                                                                     (mg/dl ± s.d.)                                                                     (mg/dl ± s.d.)                               GROUP 15 days 22 days 35 days 49 days                                         __________________________________________________________________________    Control                                                                             1266 ± 349.2                                                                       1453 ± 553.4                                                                       1570 ± 238.5                                                                       1694 ± 328.3                                 Group.sup.a                                                                   Treatment                                                                            636 ± 111.4                                                                       785.3 ± 106.7                                                                      1218 ± 85.9                                                                        1678 ± 382.5                                 Group.sup.b                                                                   __________________________________________________________________________     .sup.a 5 animals                                                              .sup.b 4 animals                                                              .sup.c s.d. refers to standard deviation                                 

The above results indicate thatbis(3,5-ditertiarybutyl-4-hydroxyphenylthio)methane reduces plasmacholesterol in cholesterol-fed rabbits at least at 15 and 22 days afterinitiating cholesterol treatment.

What is claimed is:
 1. A method of lowering total serum cholesterol in apatient in need thereof comprising administering to said patient atherapeutically effective hypocholesterolemic amount ofbis(3,5-di-tertiary-butyl-4-hydroxyphenylthio) methane.
 2. A method oftreating a patient for hypercholesterolemia comprising administering tosaid patient a therapeutically effective hypocholesterolemic amount ofbis(3,5-di-tertiary-butyl-4hydroxyphenylthio)methane.